1. Field of the Invention
The present invention relates to a method for increasing the growth rate of cells, and increasing the expression of genes in these cells, in static liquid media by using gelatinising agents in the medium, and to the use of gelatinising agents, such as xanthan, in such methods.
2. Description of the Related Art
The full citation to the references cited herein may be found in the References section below.
Suitable growth conditions for using artificial nutrient media were described a long time ago for a number of microorganisms such as bacteria or fungi, for example S. cerevisiae, B. cinerea, S. tritici, M. grisea, P. infestans or R. solani. Such artificial media are also known for mammalian cell or insect cell cultures and for plant cell cultures. These media are also used, for example, in high-throughput methods based on using cell cultures. Thus, S. cerevisiae, for example, has been successfully used in cell-based assays in HTS-experiments. However, it is likewise known that, while growth or germination on plate media is possible for a large number of different cell types, such as fungi, culture in liquid media is either not possible or very elaborate and consequently unsatisfactory (e.g. rust fungi, oomycetes). A third group of fungi, i.e. what are termed obligatory biotrophic fungi, cannot be maintained on artificial media. These are fungi which enter into a long-term nutritional relationship with the living cells of their host. In principle, the culture of cells in liquid media requires that the media be thoroughly mixed continuously, i.e. shaken, in order to ensure that the cells are supplied with nutrients. For this reason, most cells, e.g. fungal cells, are poorly suited, or not suited, for HTS methods in which the liquid media cannot be shaken and are consequently static. However, as a rule, HTS methods require the growth, or at least the survival, of the cells. In addition to this, it is as a rule necessary for a reporter gene to be expressed. The use of a high cell density is no way out in this context since a cell density which is too high renders measurements of the optical density or the fluorescence impossible. These problems will be further clarified with the aid of the following example.
In addition to the yeast fungi which have been mentioned, there are other fungi which possess outstanding growth properties both in solid media and in liquid media. A representative of this group is the phytopathogenic basidiomycete Ustilago maydis. U. maydis is described as being a dimorphic fungus. In the haploid stage, it exhibits yeast-like growth (sporidium), with a generation time of about two hours in complete medium. By contrast, the second growth form, the dikaryotic filament, does not proliferate on artificial media. For this reason, the haploid sporidia are the main form in which U. maydis is cultured. In order to achieve optimum growth, it is necessary to grow the cultures while shaking.
If, on the other hand, a U. maydis culture is transferred to a static solution, which is not constantly shaken or thoroughly mixed, such as in a 384-well MTP (MTP  microtiter plate), it is then not possible to observe any increase in the optical density (FIG. 1); consequently, no growth takes place. It is therefore not possible to grow, and use, fungal cells such as U. maydis cells in such static fluid media. This is a problem, in particular, when only an in vivo test is possible, or desired, in a particular experimental arrangement due to the specific properties of a target, of a specific enzyme test or of the measurement method employed. These targets include, inter alia, targets such as membrane proteins or receptors, and also polypeptides whose purification from a cell is difficult or associated with a loss of the activity of the polypeptide.
Furthermore, in cells which harbour a constitutively expressed reporter gene, it is either not possible to observe any increase in the expression or only possible to observe a trivial increase in the expression (FIG. 2). This has to date made it impossible to use the abovementioned fungal cells in static liquid media.
The above mentioned problems consequently have a negative effect in the high-throughput search for novel, in the case of the above mentioned example fungicidal, active compounds which belong to new active compound classes and/or have new sites of action. Sites of action, that is targets or polypeptides, or else DNAs or RNAs, which cannot, in the customary manner, be overproduced in a bacterial host, purified and used in in-vitro HTS assays are therefore only accessible with great difficulty. Consequently, screening for a number of interesting targets cannot be performed on account of technical problems. However, it is frequently precisely the use of the organisms, e.g. fungi, in which it has been possible to identify these targets which is suitable for expressing the polypeptide in question and testing it directly in vivo. However, the above-described problems have thus far prevented particular cells or organisms, such as the above mentioned fungi, from being used for this purpose.